|Year : 2016 | Volume
| Issue : 7 | Page : 166-170
Changes of CD4+ T-cell subsets after radiofrequency ablation in lung cancer and its significance
Wang Shaobin, Xiong Yu, Lin Jiatian, Chen Zaizhong, Dai Luping, Chen Junhui
Department of Minimally Invasive Intervention, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China
|Date of Web Publication||21-Feb-2017|
Department of Minimally Invasive Intervention, Peking University Shenzhen Hospital, 1120 Lianhua Road, Shenzhen 518036, Guangdong
Source of Support: None, Conflict of Interest: None
Aims: To analyze the changes of CD4+ T-cell subsets following radiofrequency ablation (RFA) in lung cancer and the impact of RFA on the antitumor immunity.
Subjects and Methods: Flow cytometry was employed to detect CD4+ T-cell subsets in the peripheral blood from 45 healthy controls and 45 lung cancer patients before and after RFA. The correlation between CD4+ T-cell subsets and the clinical characteristics was discussed by comparing the results between different groups.
Results: Compared with the healthy controls, the lung cancer patients showed a decline in Th1 cells, but higher Th2, Th17, and Treg cells (P < 0.05). Th1 cell level and Th1/Th2 cell ratio were significantly lower for stage III/IV than Stage I/II and also lower for patients with Karnofsky Performance Status (KPS) score <60 than ≥ 60 (P < 0.05). The levels of Th2, Th17, and Treg cells were much higher for patients with Stage III/IV and KPS score <60 (P < 0.05). After 2 weeks of RFA, the level of Th1 cells and Th1/Th2 cell ratio increased, while the levels of Th2, Th17, and Treg cells declined (P < 0.05). For lesions with ablation volume ≥ 70%, the level of Th1 cells and Th1/Th2 cell ratio increased, whereas the levels of Th2, Th17, and Treg cells declined dramatically (P < 0.05).
Conclusions: The level of Th1 cells declined in lung cancer patients, especially for patients with Stage III/IV and KPS score <60. After RFA, the level of Th1 cells and Th1/Th2 increased, whereas the levels of Th2, Th17, and Treg cells declined, indicating an improvement of antitumor immunity. The changes were more prominent for lesions with ablation volume ≥ 70%.
Keywords: CD4+ T-cell subsets, flow cytometry, lung cancer, radiofrequency ablation
|How to cite this article:|
Shaobin W, Yu X, Jiatian L, Zaizhong C, Luping D, Junhui C. Changes of CD4+ T-cell subsets after radiofrequency ablation in lung cancer and its significance. J Can Res Ther 2016;12, Suppl S3:166-70
|How to cite this URL:|
Shaobin W, Yu X, Jiatian L, Zaizhong C, Luping D, Junhui C. Changes of CD4+ T-cell subsets after radiofrequency ablation in lung cancer and its significance. J Can Res Ther [serial online] 2016 [cited 2021 Mar 1];12:166-70. Available from: https://www.cancerjournal.net/text.asp?2016/12/7/166/200609
| > Introduction|| |
Radiofrequency ablation (RFA) applies a radio-frequency current of 460–500 kHz to the target and directly kills the tumor cells. For lung cancer patients, the RF electrodes are inserted into the lung tumor lesions under computed tomography (CT) guidance. Polar molecules in tissue are forced to continuously realign with the oscillating electric field, leading to frictions and heat shock response. Local temperature under RFA can reach up to 90°–100° or even above 100° in several minutes, inducing thermal coagulation and necrosis of the tumor tissues. Besides, RFA can prevent metastasis by inducing vascular coagulation of the tumor.
The first application of RFA to lung cancer was reported by Marasso et al. in 1998, who used single-electrode needle coupled with chemotherapy and radiotherapy for treating bronchogenic carcinoma combined with bronchial obstruction. Dupuy et al. reported the satisfactory outcome in 2000 by using multi-electrode needle in three cases of peripheral lung cancer. In China, the first application case was reported in 2001 using RF2000 generator (Radio Therapeutics Corporation) and the multi-electrode needle in thirty lung cancer patients.
RFA for lung cancer is a local, in situ deactivation technique utilizing physical principles. The impact of RFA on immunity is an intriguing issue that attracts widespread concern. In 2006, our research team studied T-cell subsets before and after RFA for lung and liver cancers and found that for both cancers, the levels of CD3+ T-cell and CD4+ T-cell as well as the CD4+ T-cell/CD8+ T-cell ratio increased.
Four CD4+ T-cell subsets have been identified, namely, Th1, Th2, Th17, and Treg cells, each displaying distinct functions and playing an important role in mediating and regulating the antitumor immunity. Th1 cells perform the roles of activating cytotoxic T lymphocyte (CTL) and natural killer (NK) cells, inhibiting tumor angiogenesis and inducing tumor cell apoptosis., Th1 cells are pathogenic and Th2 cells are protective, and the antitumor immunity is inhibited at a higher level of Th2 cells and abnormal Th1/Th2 cell ratio. Th17 promotes tumor cell growth and tumor angiogenesis, whereas Treg cells inhibit the activities of dendritic cell (DC), CTL, and NK cells, helping the escape from immune surveillance.,
This paper focused on the difference in CD4+ T-cell subsets between lung cancer patients and healthy controls and the correlation between CD4+ T-cell subsets and clinicopathological features in lung cancer. The potential role of RFA in antitumor immunity of lung cancer patients was assessed based on the comparisons across the groups.
| > Subjects and Methods|| |
Lung cancer patients and healthy controls
Forty-five lung cancer patients were the inpatients at our department, all of which were pathologically confirmed as primary lung cancer (31 males, 14 females, aged 31–72 years old with an average of 47.5). According to the 7th edition of tumour, node, metastasis classification of lung cancer by Union for International Cancer Control (UICC) and International Association for the Study of Lung Cancer in 2009, 4, 10, 15, and 16 patients were classified as Stage I, II, III, and IV, respectively. Histologically, there were 27 cases of adenocarcinoma and 18 cases of squamous cell carcinoma; by degree of differentiation, there were 12 highly differentiated cases, 11 moderately differentiated cases, and 22 lowly differentiated cases; by position, there were 18 cases of central lung cancer and 27 cases of peripheral lung cancer. The maximum lesion size was 1.5–7.0 cm with an average of 4.4 cm. There were 35 cases with Karnofsky Performance Status (KPS) score ≥ 60 and 10 cases with KPS score <60.
An equal number of age- and gender-matched healthy controls who did not take immunoenhancement or immunodepressive drugs in the past 3 months were included in the study.
Radiofrequency ablation for lung cancer
RF1500X generator (RITA, USA) and the multi-electrode needle were used. The maximum output power of radiofrequency current was 250W. The cannula-type electrode was attached with 9 microelectrodes arranged in a radial pattern, which had the maximum diameter of 5.0 cm in full extension. The highest temperature reached 125° ±3° in the target region, and the temperature changes were monitored in real-time at five measuring points. Before RFA, the patients received endotracheal intubation and vein-inhalation mixed general anesthesia under electrocardiography monitoring. The electrode was inserted into the target lesions subcutaneously under CT guidance and the microelectrodes extended starting from the size of 2 cm with an increment of 1 cm. The output power increased gradually from 30W to 40W until reaching 250W. The ablation temperature of the target lesion was set as 90°. Complications including aerothorax, intrapulmonary hemorrhage and pleural effusion were observed.
Detection of CD4+ T-cell subsets
CT4+ T-cell subsets were detected by using flow cytometry. FACS Canto flow cytometer was purchased from Becton Dickinson (USA). Fluorescein-coupled mouse anti-human monoclonal antibodies, including CD3-PE-Cy5 (batch number: 555334), CD3-APC, CD4-PE-Cy5 (batch number: 555348), CD8-APC-Cy7 (batch number: 301016), CD25-FITC (batch number: 555431), IFN-γ-APC (batch number 341117), IL-4-PE (batch number 559333), IL-17-FITC (batch number: 512304) and FOXP3-PE (batch number: 560046), were purchased from BD Bioscience (USA).
Interferon-gamma (IFN-γ), interleukin-4 (IL-4) and IL-17 were taken as the characteristic cytokines of Th1, Th2, and Th17 cells, respectively, and FOXP3 as the specific marker of natural Treg cells. Using intracellular staining technique, the levels of IFN-γ, IL-4, IL-17, and FOXP3 were detected. Utilizing surface markers for lymphocytes, the percentages of Th1, Th2, Th17, and Treg cells in the CD4+ T-cell subsets were calculated for healthy controls and lung cancer patients.
Detections of CD4+ T-cell subsets were performed in lung cancer patients before and after 2 weeks of RFA.
Results were expressed as percentages in the form of mean ± standard deviation (x̄ ± s). Means of the two groups were compared by paired sample t-test. Intergroup comparisons were performed using Wilcoxon ranked sum nonparametric test. Statistical analyses were performed using SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA) and P < 0.05 indicated significant difference.
| > Results|| |
Comparison of CD4+ T-cell subsets between healthy controls and lung cancer patients
As compared with the healthy controls, the level of Th1 cells in peripheral blood of lung cancer patients declined, whereas the levels of Th2, Th17, and Treg cells increased with a reduction in Th1/Th2 cell ratio (P < 0.05) [Table 1].
|Table 1: Results of CD4+ T-cell subsets for lung cancer and healthy controls (x̄ ±s)|
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Correlation between CD4+ T-cell subsets and clinicopathological features of lung cancer
The level of Th1 cells and Th1/Th2 cell ratio were significantly lower for Stage III/IV lung cancer than for Stage I/II cancer (P < 0.05) and also lower for patients with KPS score <60 than for those with PKS score ≥ 60 (P < 0.05). The levels of Th2, Th17, and Treg cells were much higher for Stage III/IV lung cancer than for Stage I/II cancer (P < 0.05) and also higher for patients with KPS score < 60 than for those with KPS score ≥ 60 (P < 0.05). The two groups showed no obvious correlation between age, gender, pathological type, pathological grading, and CD4+ T-cell subsets (P > 0.05) [Table 2].
|Table 2: Relationship between CD4+ T-cell subsets and clinicopathological features (x̄ ±s) |
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Changes of CD4+ T-cell subgroups before and after radiofrequency ablation
Detection of CD4+ T-cell subgroups in 45 lung cancer patients indicated that the level of Th1 cells increased after 2 weeks of RFA, while the levels of Th2, Th17, and Treg cells declined as compared with those before RFA. The Th1/Th2 cell ratio increased, and all above changes reached a significance level (P < 0.05) [Table 3].
|Table 3: Comparison of CD4+ T-cell subsets for 45 patients with lung cancer before and after radiofrequency ablation (x̄ ±s)|
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Correlation between CD4+ T-cell subsets and efficacy of radiofrequency ablation
Among 45 lung cancer cases, 36 cases had ablation volume ≥ 70% and 9 cases had ablation volume <70%. A correlation analysis indicated that for those with ablation volume ≥ 70%, the level of Th1 cells and Th1/Th2 cell ratio increased while the levels of Th2, Th17, and Treg cells declined significantly (P < 0.05). For those with ablation volume <70%, the level of Th1 cells increased considerably (P < 0.05), while the levels of Th2, Th17, and Treg cells or Th1/Th2 cell ratio did not change significantly (P > 0.05) [Table 4].
|Table 4: Relationship between CD4+ T-cell subsets and the treatment effect (x̄ ±s)|
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Immunity is closely related to tumor occurrence and development. Antitumor immunity can inhibit tumor growth once the tumor occurs, while in turn the immunity level is impaired by tumors. Therefore, immunity has a big bearing on the final outcome of tumors. Cellular immunity is a crucial component of antitumor immunity, in which CD4+ T-cells play the antitumor role.
There was a decrease in the level of Th1 cells and Th1/Th2 cell ratio and an increase in the level of Th2, Th17, and Treg cells for lung cancer patients as compared with the normal controls. This result agreed with others.,, As indicated by one-way ANOVA, gender, age, pathological type and grading did not correlate to CD4+ T-cell subsets, but CD4+ T-cell subsets correlated to UICC staging and KPS score. Th1 cell level and Th1/Th2 cell ratio were significantly lower for Stage III/IV lung cancer than for Stage I/II and also lower for patients with KPS score <60 than for those ≥ 60 (P < 0.05). The levels of Th2, Th17, and Treg cells were much higher for Stage III/IV lung cancer than for Stage I/II and also higher for patients with KPS score <60 than for those ≥ 60 (P < 0.05). Thus as the disease progressed, the antitumor immunity of the patients deteriorated rapidly.
RFA is reputed for its minimal invasiveness, good killing effect, and repeatability and now has become an emerging solution to lung cancer. For lesions smaller than 5 cm, they can be completely deactivated by RFA for once. For larger or multiple lesions, several RFA procedures are needed. RFA is the preferred choice of treatment for Stage I/II lung cancer, patients intolerant to conventional treatments or those rejecting surgery.,,,,, Stage III lung cancer is considered to be advanced. Some patients at this stage are treated by surgical resection, while others are more fit for RFA. Stage IV lung cancer is mainly treated by systemic therapy and some patients receiving RFA can achieve a reduction in tumor load and a relief of symptoms. For Stage III/IV patients, the treatment effect can be enhanced by RFA combined with chemoradiotherapy or targeted therapy.,,,,,,,,
RFA for lung cancer has dual effects: Local improvement and addressing the disequilibrium of CD4+ T-cell subsets, by restoring normal Th1/Th2 cell ratio and normal levels of Th17 and Treg cells, especially for those with ablation volume ≥ 70%. The improved immunity following RFA for lung cancer is attributed to two reasons: One is the reduction of tumor load and the immunosuppressive factors such as transforming growth factor beta and prostaglandin E2, which is conducive to the recovery of antitumor immunity;,,,,,, the other is the release of cytokines (e.g., heat shock proteins and anti-inflammatory cytokine IL-10) and various tumor antigens triggered by RFA-induced tumor necrosis, leading to Thl predominance.,,,
Abnormalities of CD4+ T-cell subsets are related to the severity of lung cancer. The disequilibrium between CD4+ T-cell subsets can be corrected by RFA, hence determining the outcome of RFA. We believe that the changes of CD4+ T-cell subsets can be used as the predictive indicators of therapeutic effect after RFA, recurrence, and prognosis of lung cancer. The existing studies on RFA for lung cancer in this respect provide favorable evidences. Li et al. conducted a dynamic monitoring of the levels of Th1 and Th2 cells before and after RFA in thirty cases of primary liver cancer. The level of Th1 cells increased obviously after RFA, while that of Th2 cells declined; but for recurrent cases, the level of Th2 cells increased. Chen et al. determined IL-17 level in surgically resected cancer tissues from 52 cases of lung cancer at follow-up visits. According to the results of Cox's proportional hazard regression model, IL-17 that reflects the level of Th17 cells in cancer tissues was regarded as an independent prognostic factor. Lower IL-17 level predicted a lower recurrence rate and longer disease-free survival and overall survival. Fietta et al. detected the changes of Treg cells in the peripheral blood of 14 lung cancer patients receiving RFA. After 3 months of RFA, two metastatic cases showed a second increase of the level of Treg cells, while 12 emotionally stable cases maintained a low level of Treg cells. From this, it can be inferred that the decline in Treg cells and the maintenance at a low level following RFA are good predictors of long-term prognosis.
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Conflicts of interest
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[Table 1], [Table 2], [Table 3], [Table 4]